Effects Of Human Amniotic Mesenchymal Stem Cells Transplantation On The Expression Of Airway Muc5ac In A Mouse Model Of Asthma

Part One isolation,cultivation and identification of human amnion mesenchymal stem cells in vitroObjectives: To isolate and foster the human amnion mesenchymal stem cells(hAMSCs)from the amniotic membrane.the stem cells were screened and identified on the flow cytometry,and the method was acquired in vitro.Methods: To acquire healthy full-term placenta delivered by pregnant woman in the Affiliated Hospital of Zunyi Medical College under sterile technical conditions,the human amnion mesenchymal stem cells were digested and isolated by mechanical combined with trypsin-collagenase and subcultured.The morphology of P1~P3 cells was observed.The phenotype characteristics of the human amnion mesenchymal stem cells were identified by flow cytometry.Results: The human amnion mesenchymal stem cells showed the typical adherent growth,fibroblastic and long spindle shape.High-level expression of CD90,CD105,CD44 and CD73 was observed in the third generation of h AMSCs,and almost no expression of CD34,CD11 b,CD19,CD45 and HLA-DR was seen.Conclusions: The human amnion mesenchymal stem cells are successfully isolated and cultured and passaged in vitro.Part two The effects of human amniotic mesenchymal stem cells transplantation on the expression airway Muc5 ac in a mouse model of asthmaObjectives: To observe the effects of the human amnion mesenchymal stem cells vein transplantation on airway Muc5 ac in asthmatic mice.Methods: Forty female BALB/c mice(6-8W)were randomly separated into five groups as fol lowings(n=8): saline control group(NS),asthma group(AS),human amniotic mesenchym al stem cells treatment group(AS+h AMSCs),dexamethasone treatment group(AS+DEX)and human amniotic mesenchymal stem cells control group(NS+h AMSCs).The mice in AS gr oup were sensitized with subcutaneous and subcutaneous injection of chicken ovalbumin(O VA)on the 1st and 13 th days,respectively.The mice were challenged by inhalation of 10% ovalbumin solution in the closed atomized cage on the 19 th day 5d,the last excitatio n 24 h after drawing.NS groups using saline instead of ovalbumin,in the same way AS g roup;in groups AS+h AMSCs and NS+h AMSCs,the third generation of the human amnion mesenchymal stem cells suspension(1×106/ml)0.25 ml were injected through tail vein,whic h were labeled with Brdu-labeled after 18 days of the first sensitization.And the other tre atments were respectively the same as groups AS and NS.In AS+DEX group,each mouse received dexamethasone(2mg/kg)i.p.30 min before challenging.Mice were sacrificed 24 hours after the final challenge.The total number of cells and classification of irrigation w ere counted,and the level of cytokines IL-5,IL-12,IL-17 in BALF were detected by ELI SA,Brdu marked in vivo colonization rate and the human amnion mesenchymal stem cell s homing ability in lung tissue were observed by immunofluorescence staining.,lung tissue pathological changes were observed by HE staining,airway epithelial goblet cells and mucus substance changes were observed by AB-PAS,and lung tissue Muc5 ac protein expression levels were detected by western bolt.Results: Human amniotic mesenchymal stem cells were detected in lung tissues of two groups of mice,and cell colonization in group AS+h AMSCs was obvious.The total cells,percentage of Eosinophile cell,IL-5,IL-17 and the score of airway inflammatory infiltration,the relative positive staining area of airway goblet cells and mucus and the collagen deposition,Muc5 ac protein expression in group AS+h AMSCs were lower than those in group AS and higher than those in group NS,and the differences were statistically significant(P<0.01).The expression level of IL-12 was higher than that in group AS,but lower than that in group NS(P<0.01).However,there was no significant difference between the NS+h AMSCs group and the NS group(P>0.05).Conclusions: 1.Exogenous human amniotic mesenchymal stem cells can be homing to damaged lung tissues,especially in AS+h AMSCs group was significantly.2.The human amniotic mesenchymal stem cells can reduce airway inflammation and airway Muc5 ac expression in asthmatic mice.The mechanism may be that human amniotic mesenchymal stem cells play an immunomodulatory role in alleviating the infiltration of airway inflammatory cells and inhibiting the expression of IL-5,IL-17 and promote IL-12 expression,thereby inhibiting the expression of various inflammatory factors,resulting in decreased airway Muc5 ac expression.